Two-component signaling is the principal form of signal transduction in prokaryotes with distinctive examples in eukaryotes. At the end of logarithmic growth, the "transition state" Bacillus cell receives multiple signals from the environment that are simultaneously reporting conditions such as temperature, cell density, nutrition availability and oxygen tension. These signals are processed to determine the most appropriate gene expression and metabolic response for survival. The characterization of the B. subtilis Pho signal transduction network has let to the hypothesis that "The processing of the multiple signals is accomplished by regulatory networks involving multiple two-component systems that function to establish dependencies or hierarchies between systems. This overlap between regulons provides a mechanism for signal integration and signal input experienced by the organism at any one time." The proposal focuses on the B. subtilis Pho regulon signal transduction network to understand how multiple two-component systems interact to participate in a signal transduction network. The PI is planning to: 1) determine if overlap of regulons occurs at the level of transcription of the phoPR operon encoding the primary Pho two-component regulators by asking what proteins control the expression of each of the four phoPR promoters; 2) determine how two two-component regulators, ResD and PhoP, interact, such that each is essential but not sufficient for transcription of an operon encoding one of the regulatory pairs, ResD and ResE; 3) identify additional regulators proposed to assist the catalytic domain of PhoR, making that domain sufficient to control the phosphate deficiency induction of the Pho regulon; 4) determine if differences in domain interaction among response regulators explains differences in target DNA-response regulator interactions or protein oligomeric state with respect to phosphorylation.